Agonistic antibody to cd27

ABSTRACT

The invention relates to a binding compound, which binds the same epitope of human CD27 as monoclonal antibody hCD27.15, produced by hybridoma hCD27.15 which was deposited with the ATCC in on Jun. 2, 2010 under number PTA-11008. In particular the invention relates to such a binding compound of claim  1  which may comprise: an antibody heavy chain variable region which may comprise at least one CDR selected from the group consisting of SEQ ID NOs: 5, 6 and 7, or a variant of any of said sequences; and/or an antibody light chain variable region which may comprise at least one CDR selected from the group consisting of SEQ ID NOs: 8, 9 and 10, or a variant of any of said sequences.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part application of internationalpatent application Serial No. PCT/EP2011/061557 filed 7 Jul. 2011, whichpublished as PCT Publication No. WO 2012/004367 on 12 Jan. 2012, whichclaims benefit of European patent application Serial No. 10169021.2filed 9 Jul. 2010.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an isolated antibody or fragmentsthereof which bind to human CD27, polynucleotides encoding such antibodyand host cells producing said antibody. The antibody may be used tostimulate lymphocyte proliferation and/or survival, to treat cancer andto combat autoimmunity or transplant rejection. In addition, theantibody may be used as a diagnostic tool and in vitro agent to promoteproliferation and/or survival of CD27⁺ cells.

BACKGROUND OF THE INVENTION

CD27, a TNF receptor family member was identified as a membrane moleculeon human T cells (van Lier et al., 1987, J Immunol 139:1589-96).According to current evidence, CD27 has a single ligand, CD70, which isalso a TNF family member (Goodwin et al., 1993, Cell 73:447-56). CD27and CD70 have also been identified and cloned in the mouse system(Gravestein et al., 1993, Eur J Immunol 23:943-50; Tesselaar et al., JImmunol 159:4959-65).

CD27 is exclusively expressed by hematopoietic cells, in particularthose of the lymphocyte lineage, i.e. T-, B- and NK cells. In the humansystem, CD27 expression in the αβ T cell lineage is induced duringpositive selection of thymocytes and maintained in naive conventionalCD4⁺ and CD8⁺ T cells (Vanhecke et al., 1997, J Immunol 159:5973-83).Upon T cell activation via the T cell antigen receptor (TCR), CD27expression increases, in a transient manner (van Lier et al., 1987, JImmunol 139:1589-96). Next, CD27 is shed from the surface of activated Tcells and the soluble form of CD27 can be detected in the serum markerfor (chronic) T cell activation (Hintzen et al., 1991, J Immunol147:29-35). Among peripheral T cells, permanent loss of CD27 expressionresults from persistent antigenic stimulation and hallmarks terminallydifferentiated effector/memory T cells, while central memory T cellsmaintain CD27 (Baars et al., 1995, J Immunol 154:17-25; Hamann et al.,1997, J Exp Med 186:1407-18). CD27 is also expressed on human γδ T cellsand induced during thymic development (Offner F et al, 1997, J Immunol158:4634-41). Moreover, loss of CD27 expression is a hallmark ofchronically stimulated γδ T cells (Gioia C et al., 2002, J Immunol168:1484-9). Generally, CD27 is an exquisite marker for cellularactivation and differentiation stages and used as such in human clinicaldiagnostics and research.

In the mouse, CD27 was found on hematopoietic stem cells, multipotentprogenitors and common lymphoid precursors (Medina et al., 2001, NatImmunol 2:718-24; Wiesmann et al., 2000, Immunity 12:193-9).

CD27 was originally defined as a human T-cell co-stimulatory moleculethat increments the proliferative response to TCR stimulation (van Lieret al., 1987, J Immunol 139:1589-96). Presence of CD70 dictates thetiming and persistence of CD27-mediated co-stimulation. Upon immuneactivation, dendritic cells, T-, B- and NK cells transiently expressCD70, contingent upon the presence of antigen, Toll-like receptoragonists or inflammatory cytokines.

CD27 stimulation using anti-CD27 mAb CLB-CD27/1 (9F4) incremented theproliferative response of human T cells to TCR stimulation (Van Lier etal., 1987, J Immunol 139:1589-96). This was confirmed using crosslinkedanti-CD27 mAb 1A4, or transfectants expressing CD70. Conversely,antibodies directed to CD27 or CD70 could block this proliferation. BothCD4⁺ and CD8⁺ T cells responded to CD27 co-stimulation (Goodwin et al.,1993, Cell 73:447-56; Kobata et al., 1994, J Immunol 153:5422-32;Hintzen et al., 1995, J Immunol 154:2612-23). Studies in miceunambiguously support the role of CD27 as a co-stimulatory receptor fornaive CD8⁺ and CD4⁺ αβ T cells. For mouse T cells, CD27 primarilypromotes their survival upon TCR-mediated activation, but in human Tcells, it additionally promotes cell cycle entry and/or activity(reviewed in Borst et al., 2005, Curr Op Immunol 17:275-281; Nolte etal., 2009, Immunol Rev 229:216-231).

Upon its transient engagement as occurs in acute infections thattemporarily upregulate CD70, CD27 supports the generation of a CD8⁺effector T cell pool in priming organs, its maintenance at the tissueeffector site, its conversion into memory cells and its potential toexercise memory function (Hendriks et al., 2003, J Exp Med198:1369-1380; Hendriks et al. 2005, J Immunol 175:1666-75, Xiao et al,2008, J Immunol 181:1071-82). Studies with CD70 blocking antibody inmouse models support the concept that CD27-CD70 interactions can make animportant contribution to generation of CD8⁺ effector T cells, e.g.after protein immunization, virus infection and allotransplantation(Taraban et al., 2004, J Immunol 173:6542-46; Bullock and Yagita, 2005,J Immunol 174:710-17; Yamada et al., 2005, J Immunol 174:1357-1364;Schildknecht et al., 2007, Eur J Immunol 37:716-28).

Transgenic expression of CD70 in immature dendritic cells sufficed toconvert immunological tolerance to virus or tumors into CD8⁺ T cellresponsiveness upon immunization with MHC class I-restricted peptide inPBS. Likewise, agonistic soluble CD70 promoted the CD8⁺ T cell responseupon such peptide immunization (Rowley et al., 2004, J Immunol172:6039-6046) and in CD70 transgenic mice, CD4⁺ and CD8⁺ effector cellformation in response to TCR stimulation was greatly facilitated (Arenset al. 2001, Immunity 15:801-12; Tesselaar et al., 2003, Nat Immunol4:49-54; Keller et al. 2008, Immunity 29:334-346). In mouse lymphomamodels, tumor rejection was improved upon CD70 transgenesis or injectionof an activating anti-mouse CD27 antibody (Arens et al., 2003, J Exp Med199:1595-1605; French et al., 2007, Blood 109:4810-15; Sakanishi andYagita, 2010, Biochem. Biophys. Res. Comm. 393:829-835; WO 2008/051424).

Generally, CD27 expression on lymphoid cells is associated with survivalpotential. Salient examples come from human adoptive T cell therapies,in cancer and AIDS patients, where long-term persisting T cells wereselected for CD27 expression. In addition, CD70 expression ontumor-infiltrating lymphocytes was positively correlated with ananti-tumor immune response, potentially reflecting effector T cellsurvival within the tumor (Ochsenbein et al., 2004, J Exp Med200:1407-17; Huang et al., 2006, J Immunol 176:7726-35).

For conventional CD4⁺ T cells, CD27 similarly promotes primary andsecondary responses (Hendriks et al., 2000, Nat Immunol 1:433-40; Xiaoet al, 2008, J Immunol 181:1071-82). Moreover, CD27 co-stimulationfavours an IL-12 independent pathway for T helper-1 development andenables CD4⁺ T cells to provide help for memory programming of CD8⁺ Tcells (Soares et al., 2007, J Exp Med 204:1095-106; Xiao et al, 2008, JImmunol 181:1071-8). In C57BL/6 mice, CD27 stimulation is consistentlyassociated with Th1-t e CD4⁺ T cell differentiation (Arens et al. 2001,Immunity 15:801-12; Soares et al., 2007, J Exp Med 204:1095-106; Xiao etal, 2008, J Immunol 181:1071-82) and in human CD4⁺ T cells in vitro,CD27 promoted Th1 development in presence of IL-12, but had nodifferentiation-inducing effect in presence of IL-4 (van Oosterwijk etal., 2007, Int Immunol 19:713-18).

In addition, CD27 stimulation was demonstrated to promote humanregulatory T cell generation and/or function (Jacquot et al., 1997, CellImmunol 179:48-54). Amongst natural regulatory T cells in human, highCD27 expression hallmarks the cells that have the highest suppressiveactivity and the CD27 high subpopulation is preferentially amplifiedduring rapamycin treatment (Koenen et al., 2005, J Immunol 174:7573-83;Coenen et al., 2006, Blood 107:1018-23). Recent observations suggestthat the CD27⁺ Treg subpopulation can differentiate into Th17 cells(Koenen et al., 2008, Blood 112:2340-52). Interestingly, CD70⁺ Blymphoma cells were found to stimulate Treg formation and impede Th17differentiation by CD27 triggering on intratumoral T cells (Yang et al.,2007, Blood 110:2537-44; Yang et al., 2009, Cancer Res 69:5522-30).

In resting B cells, CD27 expression 1s absent, but it is induced duringB cell activation in germinal centers and in human, it is subsequentlymaintained on memory B cells and plasma cells (Agematsu et al., 2000Immunol Today 21:204-206; Jung et al., 2000, Eur J Immunol30:2437-2443). CD27 also acts as a co-stimulatory receptor on B cells.In in vitro systems with human B cells, CD27-CD70 interactionsconsistently stimulate Ig secretion (Agematsu et al., 1997, Eur JImmunol 27:2073-79; Jacquot et al., 1997, J Immunol 159:2652-7).

Human NK cells can be subdivided into two functional subsets based onCD27 expression with lack of CD27 expression identifying the matureeffector cells (Sugita et al., 1992, J Immunol 149:1199-203; Vossen etal., 2008, J Immunol 180:3739-45). Data suggest a similar co-stimulatoryrole for CD27 in NK cells as for T-cells (Takeda et al., 2000, J Immunol164; 1741-1745). The functional effect of CD27 activation on NK cellswas established by increased NK mediated killing of CD70-expressingtumor cells. CD27-mediated NK cell activation also promoted thegeneration of CD8⁺ anti-tumor immunity (Aulwurm et al., 2006, Int JCancer 11S:1728-1735; Kelly et al., 2002, Nat Immunol 3:83-90).Recently, NKT cells were shown to promote CD8⁺ T cell immunity byinduction of CD70 on dendritic cells (Taraban et al., 2008, J Immunol139:1589-96).

In addition, CD27 is highly expressed on tumor cells derived fromnon-Hodgkin's lymphomas and chronic lymphocytic leukemias (Ranheim etal., 1995, Blood 85:3556-3565; van Oers et al., 1993, Blood82:3430-3436). Soluble CD27 is used as a serum marker for lymphoidmalignancy (Van Oers et al., 1993, Blood 82:3430-6).

In the research that led to the present invention it was found that thehCD27.15 mAb stimulates the proliferation and/or survival of CD27⁺cells. Enhanced proliferation and/or survival of CD27⁺ cells forms thebasis of different therapeutic uses. Monoclonal antibodies that activateCD27 are known. Two activating anti-human CD27 antibodies have beendescribed (Van Lier et al., 1987, J. Immunol. 1987, 139:1589-96; Kobataet al., 1994, J. Immunol. 153:5422-5432). In addition, activatinganti-mouse CD27 antibodies have been described (French et al., 2007,Blood 109:4810-15; WO 2008/051424; Sakanishi and Yagita, 2010, Biochem.Biophys. Res. Comm. 393:829-835). hCD27.15 is a unique anti-humanantibody, which is, in contrast to 1A4 and 9F4 able to activate humanCD27 more effectively than its ligand CD70. These characteristics ofhCD27.15 result in a significantly increased effect on proliferation ofCD8⁺ and CD4⁺ T-cells as compared to 1A4, 9F4 and Fc-CD70.Administration of hCD27.15 alone or in combination with other agents toa human being can for example be used in the treatment of cancer.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention thus relates to binding compounds that bind to the sameepitope as hCD27.15. The hybridoma producing hCD27.15 was deposited withthe ATCC on Jun. 2, 2010 and given the deposit accession numberPTA-11008.

The invention relates to all molecules that bind the same epitope as itwas found that binding to this particular epitope stimulates theproliferation and/or survival of a CD27⁺ cell.

In one embodiment, the invention relates to binding compounds, whichbind to CD27 and may comprise:

-   -   an antibody heavy chain variable region which may comprise at        least one CDR selected from the group consisting of SEQ ID NOs:        5, 6 and 7, or a variant of any of said sequences; and/or    -   an antibody light chain variable region which may comprise at        least one CDR selected from the group consisting of SEQ ID NOs:        8, 9 and 10, or a variant of any of said sequences.

In one embodiment, the invention relates to binding compounds, whichbind to CD27 and may comprise:

-   -   an antibody heavy chain variable region which may comprise a        combination of CDRs selected from the group consisting of SEQ ID        NOs: 5, 6 and 7, SEQ ID NOs: 5 and 7, SEQ ID NOs: 6 and 7, and        SEQ ID NOs: 5 and 6, or a variant of any of said sequences;        and/or    -   an antibody light chain variable region which may comprise a        combination of CDRs selected from the group consisting of SEQ ID        NOs: 8, 9 and 10, SEQ ID NOs: 8 and 10, SEQ ID NOs: 9 and 10,        and SEQ ID NOs: 8 and 9, or a variant of any of said sequences.

In one embodiment, the invention relates to any combination of heavy andlight chain variable regions having the combinations of CDRs disclosedabove, in particular the following combinations:

-   -   SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 8, 9 and 10,    -   SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 8 and 10,    -   SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 5, 6 and 7 with SEQ ID NOs: 8 and 9    -   SEQ ID NOs: 5 and 7 with SEQ ID NOs: 8, 9 and 10,    -   SEQ ID NOs: 5 and 7 with SEQ ID NOs: 8 and 10,    -   SEQ ID NOs: 5 and 7 with SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 5 and 7 with SEQ ID NOs: 8 and 9,    -   SEQ ID NOs: 6 and 7 with SEQ ID NOs: 8, 9 and 10,    -   SEQ ID NOs: 6 and 7 with SEQ ID NOs: 8 and 10,    -   SEQ ID NOs: 6 and 7 with SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 6 and 7 with SEQ ID NOs: 8 and 9,    -   SEQ ID NOs: 5 and 6 with SEQ ID NOs: 8, 9 and 10,    -   SEQ ID NOs: 5 and 6 with SEQ ID NOs: 8 and 10,    -   SEQ ID NOs: 5 and 6 with SEQ ID NOs: 9 and 10,    -   SEQ ID NOs: 5 and 6 with SEQ ID NOs: 8 and 9.

In one embodiment, the invention relates to binding compounds, whichbind to CD27 and may comprise:

-   -   an antibody heavy chain variable region which may comprise the        CDRs of SEQ ID NOs: 5, 6 and 7, or a variant of any of said        sequences; and/or    -   an antibody light chain variable region which may comprise the        CDRs of SEQ ID NOs: 8, 9 and 10, or a variant of any of said        sequences.

In one embodiment, the binding molecule binds to CD27 and comprises:

-   -   a heavy chain variable region which may comprise the amino acid        sequence of SEQ ID NO: 3 and a light chain variable region which        may comprise the amino acid sequence of SEQ ID NO: 4.

In one embodiment, the binding compound is monoclonal antibody hCD27.15as produced by hybridoma hCD27.15 (deposit accession number PTA-11008)or a humanized version thereof.

In one embodiment the binding compound is a fragment, variant orderivative of an antibody.

According to a further aspect thereof, the invention relates to anisolated polynucleotide encoding a binding compound of the invention.The invention further relates to an expression vector which may comprisethe said polynucleotide and a host cell which may comprise theexpression vector. In one embodiment, the invention relates to theisolated polynucleotides of SEQ ID NOs 1 and 2, which encode the heavyand light chain of hCD27.15, respectively.

In one embodiment, the binding compound:

-   -   binds human CD27 with a K_(D) of about 100 nM or lower; and    -   blocks binding of human CD27 to human CD70 with an IC₅₀ of about        10 nM or lower.

In one embodiment, the invention relates to a binding compound whichcompetes for a binding epitope on human CD27 with any of the abovebinding compounds and has one or more of the following characteristics:

-   -   binds human CD27 with a K_(D) of about 100 nM or lower;    -   binds to human CD27 with about the same K_(D) as an antibody        having a heavy chain which may comprise the amino acid sequence        of SEQ ID NO: 3 and a light chain which may comprise the amino        acid sequence of SEQ ID NO: 4;    -   blocks binding of human CD27 to human CD70 with an IC₅₀ of about        10 nM or lower.

The binding compound may be any one of the following:

-   -   a chimeric antibody or a fragment thereof;    -   a human antibody or a fragment thereof;    -   a humanized antibody or a fragment thereof; or    -   an antibody fragment selected from the group consisting of Fab,        Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, bispecific mAb and a diabody.

According to another aspect thereof the invention relates to an isolatedpolynucleotide encoding the binding compound of the invention. In oneembodiment, the isolated polynucleotide comprises SEQ ID NOs 1 and 2,which encode the heavy and light chain of hCD27.15.

The invention also relates to an expression vector which may comprisethe polynucleotide and to a host cell which may comprise the expressionvector or the polynucleotide.

According to a further aspect thereof, the invention relates to a methodof producing a binding compound of the invention, which methodcomprises:

-   -   (a) culturing host cell which may comprise an expression vector        that comprises a polynucleotide encoding a binding compound of        the invention under the control of suitable regulatory sequences        in culture medium under conditions wherein the polynucleotide is        expressed, thereby producing polypeptides which may comprise the        light and heavy chain variable regions; and    -   (b) recovering the polypeptides from the host cell or culture        medium.

The invention further relates to a composition which may comprise abinding compound in combination with a pharmaceutically acceptablecarrier or diluent. Such composition in one embodiment may comprise morethan one binding compound. In one embodiment, the composition comprisesone or more other active compounds in addition to the one or morebinding compounds of the invention. Such combination compositions may beused for combination therapy, for example in the treatment of cancer. Inthat case the binding compound is combined with one or more of the usualanticancer drugs. For other combination therapies other additionalactive compounds are used. For combination therapy it is not obligatoryto have the two or more active compounds in the same composition. Thus,also part of the invention is the combined or subsequent use of thebinding compounds and the other active compound, wherein the bindingcompound and the other active compound are administered simultaneouslyor subsequently.

The invention further relates to the use of the binding compounds intherapy and diagnosis and for other, non-therapeutic purposes.

In one embodiment, the therapy comprises stimulation of proliferationand/or survival of CD27⁺ cells. In one embodiment, the therapy comprisesthe treatment of cancer. In one embodiment, the therapy comprises thetreatment of an autoimmune disease.

The binding compound of the invention when used in non-therapeuticapplications may for example be applied in techniques such asflow-cytometry, Western blotting, enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.

In the following these binding compounds will be referred to as “bindingcompounds based on hCD27.15”. This phrase is intended to encompass everycompound that binds to the epitope of CD27 recognized by hCD27.15 asdescribed above. Such compounds may be antibodies that have one or moreof the CDR regions of hCD27.15 or fragments, variants or derivativesthereof, or the monoclonal antibody hCD27.15 or a humanized versionthereof or other molecules that are capable of binding to this epitope.

In one embodiment, the therapy of the invention comprises targetingCD27⁺ CD4⁺ or CD8⁺ T cell subsets such as Tregs, Th17 cells or Th1 cellswith a binding compound based on hCD27.15, in particular the hCD27.15mAb. Targeting these CD27⁺ cells with binding compounds based onhCD27.15, in particular the hCD27.15 mAb, will direct the nature ofCD4⁺T cell cytokine production and CD4⁺ T cell help for CD8⁺ T cellresponses, which is beneficial in treating various disease situations,including cancer and auto-immunity. Examples are formed by, but notrestricted to lymphocyte derived tumors such as non-Hodgkin's lymphoma,Chronic Lymphocytic leukemias; solid tumors like pancreatic, colon andprostate carcinomas. For this purpose, hCD27.15 may be dosed directly tosubjects, alone or in combination with other anti-cancer agents.Examples of use in autoimmunity include, but are not restricted toRheumatoid Arthritis, Systemic Lupus Erythematosus and Psoriasis.

In addition, the therapy may be directed to infections, such as viraland microbial infections. Examples include, but are not restricted toadministering hCD27.15 alone or in combination with other anti-infectivedrugs to a subject who has been infected with influenza virus or CMVvirus.

In one embodiment, stimulations of the immune system with bindingcompounds based on hCD27.15, in particular the hCD27.15 mAb, may be usedto increase vaccine responses. Non-limiting examples of vaccines thatmay be used in combination with hCD27.15 stimulation include DNA-,cell-based or peptide-based vaccines that are designed to elicit a CD8⁺T cell response to cancer or infectious agents. For this purpose,binding compounds based on hCD27.15, in particular the hCD27.15antibody, may be administered before vaccination, at an appropriate timeafter vaccination, or be formulated into the vaccine.

Current state of the art technologies allow the isolation of CD27⁺ cellsand the isolation of different cellular subsets. After stimulation ofsuch isolated subsets of cells outside of the body of the patient withhCD27.15, they may subsequently be adoptively transferred to the patientor another patient. In one embodiment, the subset of cells is formed byCD27⁺ regulatory T-cells (Tregs), which may be isolated from patientssuffering from autoimmune disease and which have been shown todemonstrate superior suppressive characteristics.

The monoclonal antibody hCD27.15 promotes the proliferation and/orsurvival of CD27⁺ cells. State of the art technologies use the isolationof (subsets of) cells from a wide range of body fluids and organs. Basedon hCD27.15 stimulatory characteristics, binding compounds based onhCD27.15 may be used in in vitro cellular systems to promoteproliferation and/or survival of CD27⁺ cells. A non-limiting exampleforms Tregs, which have been demonstrated to have a short lifespan.Other examples form memory B-cells and activated T-cells.

Stimulation and proliferation of CD27⁺ cells using binding compoundsbased on hCD27.15, in particular the hCD27.15 mAb, may thus be used toincrease the Treg populations ex vivo, which may be adoptivelytransferred to the patient to suppress the hyper-activated immune systemof the patient. Such approach could be used to treat patients who sufferfrom an activated immune system. This strategy may thus for example beused to prevent transplant rejections and to treat autoimmune andinflammatory diseases.

Another example is the isolation of tumor-associated lymphocytes. Suchlymphocytes harbor anti-tumor activity, but are suppressed in activationby the tumor and its environment. Isolation of these lymphocytes,subsequent activation outside of the body using binding compounds basedon hCD27.15, in particular the hCD27.15 mAb, and adoptive transfer tothe patient is expected to deliver a proficient anti-tumor response.

The binding compounds based on hCD27.15, in particular the hCD27.15antibody, may also be applied in vivo to target CD27⁺ tumor cells. Thenon-modified binding compounds based on hCD27.15, in particular thehCD27.15 antibody, may for example be injected into patients with aCD27⁺ malignancy to elicit antibody-dependent cytotoxicity or otherimmune effector mechanisms. The binding compounds based on hCD27.15, inparticular the hCD27.15 antibody, may also be conjugated with a toxin orother appropriate drug to kill the targeted CD27⁺ tumor cells.

The binding compounds based on hCD27.15 may also be useful in diagnosticassays, e.g., for detecting expression of CD27 on specific cells,tissues, or in serum. For diagnostic applications, the binding compoundsbased on hCD27.15 typically will be labeled (either directly orindirectly) with a detectable moiety. Numerous labels are availablewhich may be generally grouped into the following categories: biotin,fluorochromes, radionucleotides, enzymes, iodine, and biosyntheticlabels.

Soluble CD27 present in the serum and other body fluids of a range ofdifferent patients has been shown to correlate with disease severity ofthe patients. For example, patients suffering from chronic lymphocyticleukemia, acute lymphoblastic leukemia and non-Hodgkin's lymphomademonstrated increased serum levels of soluble CD27. Based on thedemonstrated binding characteristics of hCD27.15, binding compoundsbased on hCD27.15 may be used as a diagnostic tool to detect solubleCD27 in the body fluids.

The binding compounds based on hCD27.15 of the present invention may beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays.Zola, Monoclonal Antibodies. A Manual of Techniques, pp. 147-158 (CRCPress, Inc. 1987).

The binding compounds based on hCD27.15 may also be used for in vivodiagnostic assays. Generally, the binding compound is labeled with aradionuclide so that the antigen or cells expressing it may be localizedusing immunoscintigraphy or positron emission tomography.

According to another aspect of the invention, the binding compounds haveother, non-therapeutic uses. The non-therapeutic uses for these bindingcompounds based on hCD27.15 include flow cytometry, western blotting,enzyme linked immunosorbant assay (ELISA) and immunohistochemistry.

The binding compounds based on hCD27.15 of this invention may forexample be used as an affinity purification reagent via immobilizationto a Protein A-Sepharose column.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

DEPOSITS

The Deposits with the ATCC, under deposit accession number PTA-11008were made pursuant to the terms of the Budapest Treaty. Upon issuance ofa patent, all restrictions upon the deposit will be removed, and thedeposit is intended to meet the requirements of 37 CFR §§1.801-1.809.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced if necessaryduring that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1. Characterization of anti-hCD27 antibody. A. Binding of hCD27.15to CHO-K₁ that were stably transfected with pCI-neo-hCD27 (CHO-K1.CD27).Anti-hCD27 57703 (R&D systems) and anti-hCD27 1A4 (Beckman Coulter) arepositive controls. Antibodies were not reactive with CHO-K1 controlcells (data not shown). B. Effect of hCD27.15 on binding of solublerecombinant hCD27-Fc fusion protein to CHO-K1 cells that had been stablytransfected with pCI-neo-hCD70 (CHO-K1.CD70). C. Effect of hCD27.15 onbinding of recombinant hCD70-mCD8 fusion protein to CHO-K1.CD27.

FIG. 2. hCD27.15 induces CD27 signaling leading to NF-κB activation.Human embryonic kidney cells (HEK293T) were transiently transfected toexpress an NF-κB-luciferase reporter construct together with a hCD27encoding vector or a control vector. The cells were stimulated for 20hours in presence or absence of hCD27.15 mAb (10 μg/ml). Stimulationwith hCD27.15 mAb revealed specific CD27-induced NF-κB activation, asread out by luciferase activity. (A) Absolute values of luciferaseactivity as read out by luciferin bioluminescence after stimulation ofhCD27-expressing HEK293T cells with mAb hCD27.15 or an isotype controlmAb. Data represent triplicate measurements from 1 experiment (+SD).Significance was measured using 2-tailed Student's t test. (B) Foldinduction of luciferase activity after stimulation of HEK293T cellstransfected to express hCD27 or control vector with hCD27.15 mAb. Datawere obtained from 3 independent experiments (N=3+SD). (C) hCD27.15 issuperior to other hCD27 agonists. HEK293T cells expressing CD27 and theNF-κB-luciferase reporter were stimulated with soluble agonisticrecombinant CD70 protein (Fc-mCD70, 2 μg/ml), mAb hCD27.15 (10 μg/ml),or equal concentrations of other mAbs directed against hCD27. Luciferaseactivity was read out at the indicated time points. Data representtriplicate measurements from 1 experiment (+SD). Significance wasmeasured using 2-tailed Student's t test.

FIG. 3. hCD27.15 induces proliferation and/or promotes survival of CD4⁺CD25⁻ T-cells. CD4⁺ CD25⁻ T-cells were isolated by MACS (neg. selection)from human PBMC's and cultured in 96 well-plates at a concentration of1×10⁵ cells/well. Stimuli were added as indicated in the Figure andproliferation was determined by [³H] thymidine incorporation.

FIG. 4. hCD27.15 induces proliferation and/or promotes survival of naïveCD8⁺ T-cells. Naïve CD8⁺ T cells were purified from human PBMC, labeledwith CFSE and stimulated for 6 days with anti-CD3 and anti-CD28 mAbs inpresence or absence of hCD27.15 mAb (10 μg/ml). hCD27.15 mAb stimulatescell division, as hallmarked by the percentage of cells in each divisioncycle (A) and total live cell yield, as hallmarked by the absolutenumber of cells in each division cycle (B). Data were obtained from 4independent experiments with cells of 4 healthy individuals (N=4+/−SEM).

FIG. 5. hCD27.15 stimulates CD8⁺ T cells to produce specific cytokinesNaïve CDS+ T cells were isolated as indicated for FIG. 3 and stimulatedwith anti-CD3 and anti-CD28 mAb in presence or absence of hCD27.15 mAb(10 μg/ml). (A) After culture for 72 hours, supernatants of cells weretaken and cytokines were measured by using Luminex assay. (B) Cellnumbers were not significantly different after 72 hours of culture,indicating a qualitative difference in the secretion of certaincytokines (A,B) Data obtained from 3 independent experiments with cellsof 3 healthy individuals (N=3+/−SEM).

FIG. 6 shows the variable region sequences of hCD27.15. Panel A and Bshow the amino acid sequences of the heavy and light chain variablesequence of hCD27.15, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” refers to any form of antibody that exhibits thedesired biological activity, such as inhibiting binding of a ligand toits receptor, or by inhibiting ligand-induced signaling of a receptor.In the present case the biological activity comprises agonist activityon CD27. Thus, “antibody” is used in the broadest sense and specificallycovers, but is not limited to, monoclonal antibodies (including fulllength monoclonal antibodies), polyclonal antibodies, and multispecificantibodies (e.g., bispecific antibodies).

“Antibody fragment” and “antibody binding fragment” mean antigen-bindingfragments and analogues of an antibody, typically including at least aportion of the antigen binding or variable regions (e.g. one or moreCDRs) of the parental antibody. An antibody fragment retains at leastsome of the binding specificity of the parental antibody. Typically, anantibody fragment retains at least 10% of the parental binding activitywhen that activity is expressed on a molar basis. Preferably, anantibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100%or more of the parental antibody's binding affinity for the target.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules, e.g., sc-Fv, unibodies (technology fromGenmab); nanobodies (technology from Domantis); domain antibodies(technology from Ablynx); and multispecific antibodies formed fromantibody fragments. Engineered antibody variants are reviewed inBolliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

An “Fab fragment” is comprised of one light chain and the CH₁ andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments which may comprise theC_(H)1 and C_(H)2 domains of an antibody. The two heavy chain fragmentsare held together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond may be formed between the two heavy chains of two Fab′fragments to form a F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “single-chain Fv antibody” (or “scFv antibody”) refers to antibodyfragments which may comprise the V_(H) and V_(L) domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the scFv to form thedesired structure for antigen binding. For a review of scFv, seePluckthun, 1994, The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. Seealso, International Patent Application Publication No. WO 88/01649 andU.S. Pat. Nos. 4,946,778 and 5,260,203.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragments may comprise a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). By using a linker thatis too short to allow pairing between the two domains on the same chain,the domains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90:6444-6448.

A “domain antibody fragment” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody fragment. The two V_(H) regions of a bivalentdomain antibody fragment may target the same or different antigens.

As used herein antibody hCD27.15 is a mouse antibody wherein the heavychain has the variable region sequence of SEQ ID NO: 3 and is joined toa IgG1 constant region and the light chain has the variable regionsequence of SEQ ID NO: 4 and is joined to the K constant region. Thehybridoma producing the hCD27.15 antibody was deposited with ATCC onJun. 2, 2010 under number PTA-11008.

An antibody fragment of the invention may comprise a sufficient portionof the constant region to permit dimerization (or multimerization) ofheavy chains that have reduced disulfide linkage capability, for examplewhere at least one of the hinge cysteines normally involved ininter-heavy chain disulfide linkage is altered as described herein. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fe region, retains at least one of the biological functions normallyassociated with the Fe region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC (antibody dependentcellular cytotoxicity) function, and/or complement binding (for example,where the antibody has a glycosylation profile necessary for ADCCfunction or complement binding).

The term “chimeric” antibody refers to antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(See, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984,Proc. Natl. Acad. Sci. USA 81:6851-6855).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody may comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso may comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The humanizedforms of rodent antibodies may essentially comprise the same CDRsequences of the parental rodent antibodies, although certain amino acidsubstitutions may be included to increase affinity, increase stabilityof the humanized antibody, or for other reasons. However, as CDR loopexchanges do not uniformly result in an antibody with the same bindingproperties as the antibody of origin, changes in framework residues(FR), residues involved in CDR loop support, might also be introduced inhumanized antibodies to preserve antigen binding affinity (Kabat et al.,1991, J. Immunol. 147:1709).

The term “antibody” also includes “fully human” antibodies, i.e.,antibodies that may comprise human immunoglobulin protein sequencesonly. A fully human antibody may contain murine carbohydrate chains ifproduced in a mouse, in a mouse cell, or in a hybridoma derived from amouse cell. Similarly, “mouse antibody” or “rat antibody” refer to anantibody that may comprise only mouse or rat immunoglobulin sequences,respectively. A fully human antibody may be generated in a human being,in a transgenic animal having human immunoglobulin germline sequences,by phage display or other molecular biological methods. Also,recombinant immunoglobulins may also be made in transgenic mice. SeeMendez et al., 1997, Nature Genetics 15:146-156. See also Abgenix andMedarex technologies.

The binding compounds of the present invention also include antibodieswith modified (or blocked) Fc regions to provide altered effectorfunctions. See, e.g. U.S. Pat. No. 5,624,821; WO2003/086310;WO2005/120571; WO2006/0057702; Presta, 2006, Adv. Drug Delivery Rev.58:640-656. Such modification may be used to enhance or suppress variousreactions of the immune system, with possible beneficial effects indiagnosis and therapy. Alterations of the Fc region include amino acidchanges (substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc may alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta, 2005, J. Allergy Clin. Immunol. 116:731 at 734-35.

The binding compounds of the present invention also include antibodieswith intact Fc regions that provide full effector functions, e.g.antibodies of isotype IgG1, which induce complement-dependentcytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC) inthe a targeted cell.

The antibodies may also be conjugated (e.g., covalently linked) tomolecules that improve stability of the antibody during storage orincrease the half-life of the antibody in vivo. Examples of moleculesthat increase the half-life are albumin (e.g., human serum albumin) andpolyethylene glycol (PEG). Albumin-linked and PEGylated derivatives ofantibodies may be prepared using techniques well known in the art. See,e.g. Chapman, 2002, Adv. Drug Deliv. Rev. 54:531-545; Anderson andTomasi, 1988, J. Immunol. Methods 109:37-42; Suzuki et al., 1984,Biochim. Biophys. Acta 788:248-255; and Brekke and Sandlie, 2003, NatureRev. 2:52-62.

Binding compounds, in particular antibodies, used in the presentinvention will usually bind with at least a KD of about 10⁻³ M, moreusually at least 10⁻⁶ M, typically at least 10⁻⁷ M, more typically atleast 10⁻⁸ M, preferably at least about 10⁻⁹ M, and more preferably atleast 10⁻¹⁰ M, and most preferably at least 10⁻¹¹ M. See, e.g. Presta,et al., 2001, Thromb. Haemost. 85:379-389; Yang, et al., 2001, Crit.Rev. Oncol. Hematol. 38:17-23; Carnahan, et al., 2003, Clin. Cancer Res.(Suppl.) 9:3982s-3990s. Antibody affinities may be determined usingstandard analysis.

The term “hypervariable region,” as used herein, refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region may comprise amino acid residues from a“complementarity determining region” or “CDR,” defined by sequencealignment, for example residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (see Kabat et al., 1991,Sequences of proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md.) and/or thoseresidues from a “hypervariable loop” (HVL), as defined structurally, forexample, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain (see Chothia and Leskl, 1987, J. Mol. Biol.196:901-917). “Framework” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies which may comprise the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations that typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al., 1975, Nature 256:495, or may be made by recombinant DNAmethods (see, for example, U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., 1991, Nature 352:624-628 andMarks et al., 1991, J. Mol. Biol. 222:581-597, for example. Themonoclonal antibodies herein specifically include “chimeric” antibodies.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells, natural killercells, myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, an “immunoconjugate” refers to an anti-CD27 antibody, ora fragment thereof, conjugated to a therapeutic moiety, such as abacterial toxin, a cytotoxic drug or a radiotoxin. Toxic moieties may beconjugated to antibodies of the invention using methods available in theart.

As used herein, a sequence “variant” refers to a sequence that differsfrom the disclosed sequence at one or more amino acid residues but whichretains the biological activity of the resulting molecule. The inventionrelates to variants of binding compounds based on hCD27.15 and tovariants of hCD27.15.

“Conservatively modified variants” or “conservative amino acidsubstitution” refers to substitutions of amino acids that are known tothose of skill in this art and may be made generally without alteringthe biological activity of the resulting molecule. Those of skill inthis art recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson, et al., Molecular Biology of theGene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)). Suchexemplary substitutions are preferably made in accordance with those setforth below as follows:

Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

As used herein, the term “about” refers to a value that is within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e. the limitations of the measurement system.For example, “about” may mean within 1 or more than 1 standard deviationper the practice in the art. Alternatively, “about” or “comprisingessentially of” may mean a range of up to 20%. Furthermore, particularlywith respect to biological systems or processes, the terms may mean upto an order of magnitude or up to 5-fold of a value. When particularvalues are provided in the application and claims, unless otherwisestated, the meaning of “about” or “comprising essentially of” should beassumed to be within an acceptable error range for that particularvalue.

“Specifically” binds, when referring to a ligand/receptor,antibody/antigen, or other binding pair, indicates a binding reactionwhich is determinative of the presence of the protein, e.g., CD27, in aheterogeneous population of proteins and/or other biologics. Thus, underdesignated conditions, a specified ligand/antigen binds to a particularreceptor/antibody and does not bind in a significant amount to otherproteins present in the sample.

“Administration”, “therapy” and “treatment,” as it applies to an animal,human, experimental subject, cell, tissue, organ, or biological fluid,refers to contact of an exogenous pharmaceutical, therapeutic,diagnostic agent, or composition to the animal, human, subject, cell,tissue, organ, or biological fluid. “Administration”, “therapy” and“treatment” may refer, e.g., to therapeutic, pharmacokinetic,diagnostic, research, and experimental methods. Treatment of a cellencompasses contact of a reagent to the cell, as well as contact of areagent to a fluid, where the fluid is in contact with the cell.“Administration”, “therapy” and “treatment” also mean in vitro and exvivo treatments, e.g., of a cell, by a reagent, diagnostic, bindingcomposition, or by another cell.

Monoclonal antibodies may be made according to knowledge and skill inthe art of injecting test subjects with human CD27 antigen and thengenerating hybridomas expressing antibodies having the desired sequenceor functional characteristics. DNA encoding the monoclonal antibodies isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the monoclonal antibodies).The hybridoma cells serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies will be described in more detail below.

Antibodies or antibody fragments may be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature, 348:552-554. Clackson et al., 1991, Nature, 352:624-628,and Marks et al., 1991, J. Mol. Biol. 222:581-597 describe the isolationof murine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity (nMrange) human antibodies by chain shuffling (Marks et al., 1992,Bio/Technology, 10:779-783), as well as combinatorial infection and invivo recombination as a strategy for constructing very large phagelibraries (Waterhouse et al., 1993, Nuc. Acids. Res. 21:2265-2266).Thus, these techniques are viable alternatives to traditional monoclonalantibody hybridoma techniques for isolation of monoclonal antibodies.

The antibody DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for non-immunoglobulin material (e.g., proteindomains). Typically such non-immunoglobulin material is substituted forthe constant domains of an antibody, or is substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody which may comprise one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

A humanized antibody has one or more amino acid residues from a sourcethat is non-human. The non-human amino acid residues are often referredto as “import” residues, and are typically taken from an “import”variable domain. Humanization may be performed generally following themethod of Winter and co-workers (Jones et al., 1986, Nature 321:522-525;Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988,Science 239:1534-1536), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are antibodies wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites innon-human, for example, rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., 1987, J.Immunol. 151:2296; Chothia et al., 1987, J. Mol. Biol. 196:901). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285;Presta et al., 1993, J. Immunol. 151:2623).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues may beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Humanization of antibodies is a straightforward protein engineeringtask. Nearly all murine antibodies may be humanized by CDR grafting,resulting in the retention of antigen binding. See, Lo, Benny, K. C.,editor, in Antibody Engineering: Methods and Protocols, volume 248,Humana Press, New Jersey, 2004.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovitset al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year inImmunology 7:33; and Duchosal et al., 1992, Nature 355:258. Humanantibodies may also be derived from phage-display libraries (Hoogenboomet al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991,222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

Amino acid sequence variants of humanized anti-CD27 antibodies areprepared by introducing appropriate nucleotide changes into thehumanized anti-CD27 antibodies' DNAs, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions into,and/or substitutions of, residues within the amino acid sequences shownfor the humanized anti-CD27 antibodies. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe humanized anti-CD27 antibodies, such as changing the number orposition of glycosylation sites.

A useful method for identification of certain residues or regions of thehumanized anti-CD27 antibodies polypeptides that are preferred locationsfor mutagenesis is called “alanine scanning mutagenesis,” as describedby Cunningham and Wells, 1989, Science 244:1081-1085. Here, a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, H is, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with CD27 antigen. The amino acidresidues demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, Ala scanning or random mutagenesis isconducted at the target codon or region and the expressed humanizedanti-CD27 antibodies' variants are screened for the desired activity.

Ordinarily, amino acid sequence variants of the humanized anti-CD27antibodies will have an amino acid sequence having at least 75% aminoacid sequence identity with the original mouse antibody amino acidsequences of either the heavy or the light chain more preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,and most preferably at least 95%, 98% or 99%. Identity or homology withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with thehumanized residues, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. The percentage of identitybetween two sequences may be determined with computer application suchas SeqMan II (DNAstar Inc, version 5.05). Using this program twosequences may be aligned using the optimal alignment algorithm of Smithand Waterman (1981) (Journal of Molecular Biology 147:195-197). Afteralignment of the two sequences the percentage identity may be calculatedby dividing the number of identical nucleotides between the twosequences by the length of the aligned sequences minus the length of allgaps.

Antibodies having the characteristics identified herein as beingdesirable in humanized anti-CD27 antibodies may be screened forincreased biologic activity in vitro or suitable binding affinity. Toscreen for antibodies that bind to the epitope on human CD27, a routinecross-blocking assay such as that described in Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988),may be performed. Antibodies that bind to the same epitope are likely tocross-block in such assays, but not all cross-blocking antibodies willnecessarily bind at precisely the same epitope since cross-blocking mayresult from steric hindrance of antibody binding by antibodies atoverlapping epitopes, or even nearby non-overlapping epitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.,1995, J. Biol. Chem. 270:1388-1394, may be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells, 1989, Science244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human CD27 may also be used to determine the functionalepitope for anti-CD27 antibodies of the present invention. Additionalantibodies binding to the same epitope as an antibody of the presentinvention may be obtained, for example, by screening of antibodiesraised against CD27 for binding to the epitope, or by immunization of ananimal with a peptide which may comprise a fragment of human CD27 whichmay comprise the epitope sequences. Antibodies that bind to the samefunctional epitope might be expected to exhibit similar biologicalactivities, such as blocking receptor binding, and such activities maybe confirmed by functional assays of the antibodies.

Antibody affinities may be determined using standard analysis. Preferredbinding compounds such as e.g. humanized antibodies are those that bindhuman CD27 with a Kd value of no more than about 1×10⁻⁷; preferably nomore than about 1×10⁻⁸; more preferably no more than about 1×10⁻⁹; andmost preferably no more than about 1×10⁻¹⁰ or even 1×10⁻¹¹ M.

The humanized antibody may be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, theantibody is an IgG antibody. Any isotype of IgG may be used, includingIgG1, IgG2, IgG3, and IgG4. Variants of the IgG isotypes are alsocontemplated. The humanized antibody may comprise sequences from morethan one class or isotype. Optimization of the necessary constant domainsequences to generate the desired biologic activity is readily achievedby screening the antibodies in the biological assays described in theExamples.

Likewise, either class of light chain may be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

The antibodies and antibody fragments of the invention may also beconjugated with cytotoxic payloads such as cytotoxic agents orradionucleotides such as, ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I,¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu,⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and, ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn,⁵²Tr and, ⁵⁶Fe. Such antibody conjugates may be used in immunotherapy toselectively target and kill cells expressing a target (the antigen forthat antibody) on their surface. Exemplary cytotoxic agents includericin, vinca alkaloid, methotrexate, Psuedomonas exotoxin, saporin,diphtheria toxin, cisplatin, doxorubicin, abrin toxin, gelonin andpokeweed antiviral protein.

The antibodies and antibody fragments of the invention may also beconjugated with fluorescent or chemiluminescent labels, includingfluorophores such as rare earth chelates, fluorescein and itsderivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

Any method known in the art for conjugating the antibody molecules orprotein molecules of the invention to the various moieties may beemployed, including those methods described by Hunter et al., 1962,Nature 144:945; David et al., 1974, Biochemistry 13:1014; Pain et al.,1981, J. Immunol. Meth. 40:219; and Nygren, J., 1982, Histochem. andCytochem. 30:407. Methods for conjugating antibodies and proteins areconventional and well known in the art.

When using recombinant techniques, the antibody may be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., 1992, Bio/Technology 10:163-167 describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris may be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells may be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc region that is present in the antibody. Protein A maybe used to purify antibodies that are based on human Ig.gamma1,Ig.gamma2, or Ig.gamma4 heavy chains (Lindmark et al., 1983, J. Immunol.Meth. 62:1-13). Protein G is recommended for all mouse isotypes and forhuman .gamma.3 (Guss et al., 1986, EMBO J 5:1567-1575). The matrix towhich the affinity ligand is attached is most often agarose, but othermatrices are available.

Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl) benzene allow for faster flow rates and shorterprocessing times than may be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In one embodiment, the glycoprotein may be purified using adsorptiononto a lectin substrate (e.g. a lectin affinity column) to removefucose-containing glycoprotein from the preparation and thereby enrichfor fucose-free glycoprotein.

The invention may comprise pharmaceutical formulations of a CD27 bindingcompound. To prepare pharmaceutical or sterile compositions, the bindingcompound, in particular an antibody or fragment thereof, is admixed witha pharmaceutically acceptable carrier or excipient, see, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984). Formulations oftherapeutic and diagnostic agents may be prepared by mixing withphysiologically acceptable carriers, excipients, or stabilizers in theform of, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al., 2001, Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro, 2000, Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.),1993, Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990,Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Toxicity and therapeutic efficacy of the binding compound, in particularantibody, compositions, administered alone or in combination withanother agent, such as the usual anti-cancer drugs, may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it may be expressed as the ratio between LD₅₀and ED₅₀. The data obtained from these cell culture assays and animalstudies may be used in formulating a range of dosage for use in humans.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

Suitable routes of administration include parenteral administration,such as intramuscular, intravenous, or subcutaneous administration andoral administration. Administration of binding compounds such asantibodies, used in the pharmaceutical composition or to practice themethod of the present invention may be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral,intraarterial or intravenous injection. In one embodiment, the bindingcompound of the invention is administered intravenously. In anotherembodiment, the binding compound of the invention is administeredsubcutaneously.

Alternatively, one may administer the binding compound in a local ratherthan systemic manner, for example, via injection of the binding compounddirectly into the site of action, often in a depot or sustained releaseformulation. Furthermore, one may administer the antibody in a targeteddrug delivery system.

Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak, 1996, AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991,Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003,New Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med.341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792;Beniaminovitz, et al., 2000, New Engl. J. Med. 342:613-619; Ghosh, etal., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl.J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

A preferred dose protocol is one involving the maximal dose or dosefrequency that avoids significant undesirable side effects. A totalweekly dose is generally at least 0.05 μg/kg body weight, more generallyat least 0.2 μg/kg, most generally at least 0.5 μg/kg, typically atleast 1 μg/kg, more typically at least 10 μg/kg, most typically at least100 μg/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0mg/kg, most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg,more optimally at least 25 mg/kg, and most optimally at least 50 mg/kg(see, e.g., Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold,et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J.Neural. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, CancerImmunol. Immunother. 52:133-144). The desired dose of a small moleculetherapeutic, e.g., a peptide mimetic, natural product, or organicchemical, is about the same as for an antibody or polypeptide, on amoles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with diseaseand/or a reduction in the severity of such symptoms that will or areexpected to develop with said disease. The terms further includeameliorating existing symptoms, preventing additional symptoms, andameliorating or preventing the underlying causes of such symptoms. Thus,the terms denote that a beneficial result has been conferred on avertebrate subject with a disease.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of an anti-CD27 antibody orfragment thereof, that when administered alone or in combination with anadditional therapeutic agent to a cell, tissue, or subject is effectiveto prevent or ameliorate the disease or condition to be treated. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously. An effective amount oftherapeutic will decrease the symptoms typically by at least 10%;usually by at least 20%; preferably at least about 30%; more preferablyat least 40%, and most preferably by at least 50%.

Methods for co-administration or treatment with a second therapeuticagent are well known in the art, see, e.g., Hardman, et al. (eds.),2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics,10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.), 2001,Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.),2001, Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., PA.

The pharmaceutical composition of the invention may also contain otheragents, including but not limited to a cytotoxic, chemotherapeutic,cytostatic, anti-angiogenic or antimetabolite agents, a tumor targetedagent, an immune stimulating or immune modulating agent or an antibodyconjugated to a cytotoxic, cytostatic, or otherwise toxic agent. Thepharmaceutical composition may also be employed with other therapeuticmodalities such as surgery, chemotherapy and radiation.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Immunization and Selection of Anti-CD27 Antibodies

Immunization of Mice with CD27 cDNA

To isolate antibodies against the human CD27 protein that harbouragonistic activity we hypothesized to find such antibodies among a setof anti-CD27 antibodies, which bind to the ligand binding site. Togenerate anti-hCD27 antibodies, the cDNA encoding the full length openreading frame of hCD27 was subcloned into the pCI-neo vector (Promega,Madison, Wis.). Expression of the obtained vector was checked bytransient transfection of pCI-neo-hCD27 in CHO-K1 cells (American TypeCulture Collection, Manassas, Va.) and flow cytometry using 10 μg/mlmouse anti-hCD27 IgG1 (BD Pharmingen #555439), followed by goatanti-mouse IgG1-FITC (1:100) (Southern Biotechnology, Birmingham, Ala.).

Mice were immunized by gene gun immunization using a Helios Gene gun(BioRad, Hercules, Calif.) and DNA coated gold bullets (BioRad)following manufacturer's instructions. Briefly, 1 μm gold particles werecoated with pCI-neo-hCD27 cDNA and commercial expression vectors formouse Flt3L and mouse GM-CSF in a 2:1:1 ratio (both from Aldevron,Fargo, N. Dak.). A total of 1 μg of plasmid DNA was used to coat 500 μgof gold particles.

Specifically, 7-8 weeks old female BALB/C mice were immunized in theears with a gene gun, receiving 3 cycles of a shot in both ears.Approximately, a 1:4,000 anti-hCD27 titer was detected by cell-ELISA inmouse serum after two DNA immunizations. In the cell-ELISA, allincubation steps were followed by a wash step with PBST (PBS with 0.01%Tween 20). Parental CHO-K1 or CHO-K1.hCD27 cells were seeded (40,000cells/well) in tissue culture plates and incubated overnight at 37° C.The next day, culture medium was removed and cells were incubated for 1hour with (dilutions of) mouse serum at 37° C. Next, cells were washedwith PBST and incubated for 1 hour at 37° C. with 1:1,000goat-anti-mouse IgG-HRP (Southern Biotechnology, #1030-05).

Subsequently, cells were washed 6 times with PBST and anti-hCD27immunoreactivity was visualized with 100 μl OptiEIA TMB substrate (BDBiosciences, Franklin Lake, N.J.). Reactions were stopped with 100 μl0.5 M H₂SO₄ and absorbances were read at 460 and 620 nm. Mice thatdemonstrated reactivity against hCD27 were immunized for a final, fourthtime and sacrificed four days later.

Erythrocyte-depleted spleen cell populations were prepared as describedpreviously (Steenbakkers et al., 1992, J. Immunol. Meth. 152:69-77;Steenbakkers et al., 1994, Mol. Biol. Rep. 19:125-134) and frozen at−140° C.

Selection of Anti-hCD27 Antibody Producing B Cells

To select B cell clones producing anti-hCD27 antibodies, 1.5×10⁷erythrocyte-depleted splenocytes were depleted for monocytes.hCD27-specific B-cells were selected by binding on irradiated (3,000RAD) CHO-K1.hCD27 transfectants, which had grown to confluency in aT25-flask. After extensive washing to delete non-specific B-cells, boundB-cells were collected by Trypsin treatment according to themanufacturer's instructions (Invitrogen, cat. no. 25200-056). Next,B-cells were cultured as described by Steenbakkers et al., 1994, Mol.Biol. Rep. 19:125-134. Briefly, selected B-cells were mixed with 7.5%(v/v) T-cell supernatant and 50,000 irradiated (2,500 RAD) EL-4 B5nursing cells in a final volume of 200 μl DMEM F12/P/S/10% BCS in96-well flat-bottom tissue culture plates.

On day eight, supernatants were screened for hCD27 reactivity bycell-ELISA as described above. Thirteen hCD27-reactive supernatants wereidentified and tested for their ability to inhibit the interactionbetween hCD27 and hCD70. In the cell-ELISA, all incubation steps werefollowed by a wash step with PBST (PBS with 0.01% Tween 20). ParentalCHO-K1 or CHO-K1.hCD27 cells were seeded (40,000 cells/well) in tissueculture plates and incubated overnight at 37° C. The next day, culturemedium was removed and cells were incubated for one hour with (dilutionsof) mouse serum at 37° C. Next, cells were washed with PBST andincubated for one hour at 37° C. with 1:1,000 goat-anti-mouse IgG-HRP(Southern Biotechnology, #1030-05). Subsequently, cells were washed 6times with PBST and anti-hCD27 immunoreactivity was visualized with 100μl TMB Stabilized Chromagen (Invitrogen, cat. no. 5B02). Reactions werestopped with 100 μl 0.5 M H₂SO₄ and absorbances were read at 460 and 620nm.

In addition, blocking properties of the supernatants were studied usingtwo competition assays. The CHO-K1.CD27 assay works along the followingprinciples: CHO-K1.CD27 cells were seeded (40,000 cells/well) in a96-well plate and incubated overnight at 37° C. After medium removal, 50μl recombinant hCD70 (CD70 (h)-muCD8 fusion Protein (Ancell, cat. no.ANC-537)) (0.5 μg/ml) and 50 μl anti-hCD27 antibody containingsupernatant were added. After 1 hour incubation at room temperature, thewells were washed three times with PBST. Next, 100 μl/wellStreptavidin-HRP conjugate (BD Pharmingen, cat. no. 554066) (1:5,000)was added and cells were incubated for 1 hour at 37° C. After 6 finalwashes with PBST the ELISA was developed as outlined above. Positivecontrols: anti-hCD27, clone 57703 (R&D systems, cat. no. MAB382) andanti-hCD27, clone 1A4 (Beckman Coulter, cat. no. IM2034). TheCHO-K1.CD70 assay works along the following principles: CHO-K1.CD70cells were seeded in a 96 well plate at a density of 40,000 cells/well.The same amount of plates was blocked by adding 300 μl medium/well, andall plates were incubated overnight at 37° C. The following day, themedium-only containing plates were emptied by flicking the plate, and 50μl/well recombinant soluble hCD27-Fc fusion protein (rhCD27/Fc chimera(0.5 μg/ml) (R&D systems cat. no. 382-CD)) was added. To these plates,50 μl antibody containing medium/sera or medium was added. After 1 hourincubation at room temperature, the 100 μl rhCD27-Fc/antibody mix wastransferred to the CHO-K1/CD70 plate(s) from which the medium had beenremoved. These plate(s) were incubated for 1 hour at room temperatureand then washed three times with PBST. 100 μl anti-human Ig (H+L)-HRPconjugate (1:2,500) was added to every well (Promega, cat. no. W4031)and the cells were incubated for 1 hour at 37° C. After 6 final washeswith PBST, the ELISA was developed as outlined above.

Positive controls: anti-hCD27, clone 57703 (R&D systems, cat. no.MAB382) and anti-hCD27, clone 1A4 (Beckman Coulter, cat. no. IM2034).

All supernatants demonstrated to contain antibodies that blocked theinteraction between hCD27 and hCD70. Subsequently, the B-cell clonesfrom the hCD27 reactive supernatants were immortalized bymini-electrofusion following published procedures (Steenbakkers et al.,1992, J. Immunol. Meth. 152:69-77; Steenbakkers et al., 1994, Mol. Biol.Rep. 19:125-34). Specifically, B-cells were mixed with 10⁶ Sp2/0-Ag14myeloma cells, and serum was removed by washing with DMEM F12 media.Cells were treated with Pronase solution (Calbiochem, cat. no.4308070.536) for 3 minutes and washed with Electrofusion Isomolar Buffer(Eppendorf, cat. no. 53702). Electrofusions were performed in a 50 μlfusion chamber by an alternating electric field of 30 s, 2 MHz, 400 V/cmfollowed by a square, high field pulse of 10 μs, 3 kV/cm and again by analternating electric field of 30 s, 2 MHz, 400 V/cm.

Contents of the chamber were transferred to hybridoma selective mediumand plated in a 96-well plate under limiting dilution conditions. On day12 following the fusions, hybridoma supernatants were screened for hCD27reactivity and hCD70-blocking activity, as described above. Sevenhybridomas secreting antibodies in the supernatant which recognizedhCD27 and demonstrated blocking activity were isolated and subcloned bylimited dilution to safeguard their integrity. Antibody hCD27.15 wasselected for further analysis.

Example 2 Purification and Characterization of Anti-hCD27 Antibodies

Stabilization of Anti-hCD27 Producing Hybridomas and Purification ofAnti-hCD27 antibodies

Clonal cell populations were obtained for the hCD27.15 hybridoma by tworounds of limiting dilutions. Stable hybridomas were cultured inserum-free media for 7-10 days; supernatants were harvested and filteredthrough a 0.22 μM nitrocellulose membrane. Antibodies were purifiedusing Prosep A spin columns according to the manufacturer's instructions(Millipore, cat. no. LSK2ABA60). Buffer was exchanged for PBS usingPD-10 gel-filtration columns (GE Healthcare). Antibodies wereconcentrated with Amicon Ultra-15 centrifugal filter units (Millipore,Billerica, Mass.) and quantified using spectrophotometry. Using a mousemonoclonal antibody isotyping test kit (Roche, #11493027001), the(sub)-isotype of all hCD27 antibodies was determined to be IgG1, Kappa.

Binding Analysis

Cell-based ELISA experiments using purified hCD27 antibodies wereperformed to determine binding activities of hCD27 to cellularlyexpressed hCD27. In this cell-ELISA, all incubation steps were followedby a wash step with PEST (PBS with 0.01% Tween 20). CHO-K1.hCD27 cellswere seeded (40,000 cells/well) in tissue culture plates and incubatedovernight at 37° C. The next day, culture medium was removed and cellswere incubated for one hour with (dilutions of) purified antibodies at37° C. Next, cells were washed with PBST and incubated for one hour at37° C. with 1:1,000 goat-anti-mouse IgG-HRP (Southern Biotechnology,#1030-05). Subsequently, cells were washed 6 times with PBST andanti-hCD27 immunoreactivity was visualized with 100 μl TMB StabilizedChromagen (Invitrogen, cat. no. 5B02). Reactions were stopped with 100μl 0.5 M H₂SO₄ and absorbances were read at 460 and 620 nm. As shown inFIG. 1A, the different hCD27 antibodies (hCD27.15 and controls) bound tohCD27 with different binding strengths. Calculated EC₅₀, representingthe concentration at which 50% of the total binding signal is observedare represented in Table 1.

TABLE 1 Overview of KD, EC₅₀ and IC₅₀ values of hCD27.15 and 1A4CD27(control). KD × IC₅₀ (ng/ml) IC₅₀ (ng/ml) 1E−9 (M) EC₅₀ (ng/ml)CHO-K1.CD27 CHO-K1.CD70 hCD27.15 122 686.5 864 1546 1A4CD27 33 93.7 370

Blocking properties of the purified antibodies were studied using twocompetition assays. The CHO-K1.CD70 assay works along the followingprinciples: CHO-K1.CD70 cells were seeded in a 96 well plate at adensity of 40,000 cells/well. The same amount of plates was blocked byadding 300 μl medium/well, and all plates were incubated overnight at37° C. The following day, the medium-only containing plates were emptiedby flicking the plate, and 50 μl/well rhCD27-Fc chimera (0.5 μg/ml) (R&Dsystems cat. no. 382-CD) was added. To these plates, 50 μl of differentdilutions of purified hCD27.15 antibodies were added. After 1 hourincubation at room temperature, the 100 μl rhCD27Fc/antibody mix wastransferred to the CHO-K1/CD70 plate(s) from which the medium had beenremoved. These plate(s) were incubated for 1 hour at room temperatureand then washed 3 times with PBST. 100 μl anti-human Ig (H+L)-HRPconjugate (1:2,500) was added to every well (Promega, cat. no. W4031)and the plates were incubated for 1 hour at 37° C. After 6 final washeswith PBST TMB Stabilized Chromagen (Invitrogen, cat. no. SB02) (100μL/well) was added and the ELISA was read out as outlined above.Positive controls: anti-hCD27, clone 57703 (R&D systems, cat. no.MAB382) and anti-hCD27, clone 1A4 (Beckman Coulter, cat. no. IM2034). Asshown in FIG. 1B, the purified hCD27.15 antibody blocked the binding ofrhCD27Fc chimera to CHO-K1.CD70 cells. Calculated IC₅₀ values ofhCD27.15 and the positive control 1A4, which represent the concentrationat which half of the inhibition is observed, are presented in Table 1.

The CHO-K1.CD27 assay works along the following principles: CHO-K1.CD27cells were seeded (40,000 cells/well) in a 96-well plate and incubatedovernight at 37° C. After medium removal, 50 μl recombinant mouse CD70fusion Protein (Fc-mCD70) (0.5 μg/ml) and 50 μl of different dilutionsof purified anti-hCD27 antibodies were added. Fc-mCD70 is a fusionprotein of murine CD70 (aa 41-195) fused at the C-terminus of thedimerization domain of human IgG1. A cDNA construct encoding this fusionprotein was constructed as described by Rowley and Al-Shamkhani, 2004, JImmunol 15:172:6039-46 and used to produce Fc-mCD70 protein in 293Thuman embryonic kidney cells. The protein was purified by affinitychromatography on Protein A Sepharose (GE Health Care).

After 1 hour incubation at room temperature, the wells were washed 3times with PBST. Next, 100 μl/well Streptavidin-HRP conjugate (BDPharmingen, cat. no. 554066) (1:5,000) was added and cells wereincubated for one hour at 37° C. After 6 final washes with PBST TMBStabilized Chrornagen (Invitrogen, cat. no. SB02) (100 μl/well) wasadded. The reaction was stopped by the addition of 100 μl 0.5 M H₂SO₄.Absorbencies were read at 460 and 620 nn. Positive controls: anti-hCD27,clone 57703 (R&D systems, cat. no. MAB382) and anti-hCD27, clone 1A4(Beckman Coulter, cat. no. IM2034). As shown in FIG. 1C, hCD27.15antibodies blocked the interaction between recombinant human CD70 andCHO-K¹.CD27 cells. Calculated IC₅₀ values are presented in Table 1.

Kinetic Analysis by Label-Free Surface Plasma Resonance (Biacore)

The binding properties of hCD27.15 antibodies were characterized in moredetail using label-free surface plasma resonance using Biacore 2000equipment. Low amounts of antibodies were coupled to a CM5 sensor chipusing amine coupling at pH=4.5, with R_(max) not exceeding 100 RU. Thiswill, in combination with a high flow level (30 μl/min) yield good fitsto the 1:1 Langmuir binding model. A concentration series of rhCD27Fcchimera, ranging from 0.016 nM to 1 nM, was injected for 1 minute at 30μl/min. The dissociation was monitored for 5 minutes. The running bufferis HEPES-buffered saline with 3 mM EDTA and 0.005% P20 (HBS-EP), pH 7.4.Combination plots were made by subtraction of the signal obtained at theblank flow cell, using BIAeval 3.2. The sensor grams were fitted to a1:1 Langmuir binding model. Antibody hCD27.15 shows a fast associationand dissociation, resulting in a moderate affinity. The calculated K_(D)values are presented in Table 1.

Species Cross-Reactivity

Binding of hCD27 antibodies to mouse CD27 was determined using MCF-7breast carcinoma cells that had been retrovirally transduced to stablyexpress the full length cDNA encoding human CD27 or mouse CD27. Emptyvector-transduced cells served as a control. Binding of the antibodieswas tested by flow cytometric analysis, with validated agonisticanti-hCD27 antibodies 1A4 and CLB CD27/1; and anti-mouse CD27 LG.3A10(Gravestein et al., 1995, Int Immunol. 7:551-7) as positive controls.These commercially available anti-hCD27 antibodies, which have beenreported to harbor agonistic activities, were obtained as described inTable 2.

TABLE 2 commercially available agonistic anti-CD27 antibodies. AntibodyCompany Cat no. 9F4 (CLB-CD27/1) Pelicluster M1455 1A4 Beckman CoulterIM2034

The hCD27.15 antibodies bound to human CD27, but not to mouse CD27 asexpressed on the MCF-7 cells.

The binding site of the hCD27.15 antibody was characterized and comparedwith the commercially available agonistic antibodies 9F4 and 1A4 using across-competition Biacore assay. Using common amine coupling at pH 4.5,flow cells were immobilized at 25 μg/ml of each antibody to a highimmobilization level. Next, multi flow cell injections of 1 nM ofrhCD27Fc chimera (R&D systems, cat. no. 382-CD) with a speed of 5 μl/minwere followed by 10 nM of the second antibody. The anti-hCD27 antibodyhCD27.15 of the invention and the two known agonistic antibodies (1A4and 9F4) were used as a primary (immobilized) or secondary (free)antibody. Agonistic anti-CD27 antibody 1A4 was only used as a secondantibody and was not immobilized because of the presence of BSA in thebuffer. Fc-mCD70 is a fusion protein of murine CD70 (aa 41-195) fused atthe C-terminus of the dimerization domain of human IgG1. A cDNAconstruct encoding this fusion protein was constructed as described byRowley and Al-Shamkhani, 2004, J Immunol 15:172:6039-46 and used toproduce Fc-mCD70 protein in 293T human embryonic kidney cells. Theprotein was purified by affinity chromatography on Protein A Sepharose(GE Health Care).

All flow cells were regenerated by a 6-second injection of 10 mM HCl at50 μl/min. An increase in signal upon injection of the second antibodymeans that the second antibody can still bind and that the primary andsecondary bind to different binding sites. If not, it suggests that bothantibodies recognize the same epitope of hCD27, have overlappingepitopes, or can not bind at the same time due to steric hindrance. Asshown in Table 3 the hCD27.15 and control antibodies can be divided intodifferent binding groups.

TABLE 3 Cross-competition assay using Biacore to determine binding sitesof the different anti-hCD27 antibodies (including agonistic antibodies1A4 and 9F4) and Fc-mCD70. First, the different anti-hCD27 antibodieswere immobilized on the CMS chip (referred to as ‘immobilized’), afterbinding of rhCD27-Fc chimera binding (R&D systems, cat. no. 382-CD) asecond antibody was injected (referred to as ‘free’). Based on thischeckerboard analysis the antibodies were divided in seven (A-G) epitopegroups. immobilized free hCD27.15 9F4 1A4 Fc-mCD70 hCD27.15 − − − 9F4− + − 1A4 n.d. n.d. n.d. Fc-mCD70 + + − ‘+’ indicates simultaneousbinding, while ‘−’ indicated binding of the second antibody is notpossible after capture by the first antibody. nd indicates, notdetermined.

Example 3 Functional Profiling of Mouse Anti-Human CD27 Antibodies

hCD27.15 Induces CD27 Signaling Leading to NF-KB Activation

The full length human CD27 cDNA was cloned into the pcDNA3 expressionvector and transiently expressed by transfection into HEK293T humanembryonic kidney cells (HEK293T), using FuGENE6 transfection reagent(Roche). CD27 expression from this construct was validated by flowcytometry. To read out hCD27 signaling in response to binding ofhCD27.15 mAb, HEK293 cells were transiently co-transfected with thehCD27 pcDNA vector or empty control vector and an NF-κB-luciferasereporter construct, encoding the luciferase gene driven by a minimalNF-κB-responsive promoter (Bonehill et al., 2008, Mol Ther16(6):1170-80). At 20 h after transfection, the cells were stimulatedfor 4, 8, 20 or 24 hours in presence or absence of hCD27.15 mAb (10μg/ml), other CD27 antibodies (described in Table 2, 10 μg/ml) or FcCD70(2 μg/ml). After stimulation, cells were washed with ice cold PBS andlysed with Cell Culture Lysis buffer (Promega, Luciferase assay system,catalog number E1500). Luciferase activity was measured after substratewas added to cell lysates following protocol of manufacturer(Luminometer Centro XS3 LB 960, Berthold Technologies). Data wasanalysed using Mikrowin 2000 software. FIG. 2 illustrates that hCD27.15activating CD27 more potently than other hCD27 antibodies and FcCD70.

hCD27.15 Costimulates Human CD4⁺CD25⁻ T Cells

The effect of activating CD27 with the hCD27.15 antibody on naïve humanCD4⁺CD25⁻ T cells was determined as follows. PBMCs were isolated fromBuffy coat using Ficoll gradient centrifugation according to themanufacturer's instruction (Ficoll-Paque™ Plus cat. Number 17-1440-03).Untouched CD4⁺CD25⁻ T-cells were isolated from these PBMC's by MACSbased negative selection using the CD4⁺ T-cell isolation kit II(Miltenyi cat. No 130-091-155) and CD25 microbeads II (Miltenyi cat. No130-092-983) according to the manufacturer's instructions. PurifiedCD4⁺CD25⁻ cells were seeded in 96 well-plates at a concentration of1×10⁵ cells/well. Prior to culturing CD4⁺CD25⁻ cells were checked forpurity by flow cytometry. Cells were incubated with differentcombinations of anti-CD3 (OKT-3), anti-CD28 (1 μg/ml:clone 15E8,Sanquin), Fc-CD70 (2 μg/ml), isotype control (MOPC-21, 10 μg/ml) andhCD27.15 (10 m/ml), as indicated for FIG. 3. The next day, proliferationwas detected by [³H] thymidine incorporation. hCD27.15 stimulated theproliferation of human CD4⁺CD25⁻ cells under suboptimal stimulationconditions.

hCD27.15 Costimulates Human CD8⁺ T Cells

The effect of activating CD27 by the hCD27.15 antibody on naïve humanCD8⁺ T cells was examined as follows. PBMCs were isolated from buffycoat using Ficoll gradient centrifugation. Untouched naïve CD8⁺ T-cellswere isolated from these PBMC by MACS-based negative selection using theBD IMag™ human naïve CD8⁺ T cell enrichment kit (BD cat number 558569),according to the manufacturer's instructions. The CD8⁺ T cells selectedwere checked for purity and naïvety by flow cytometry using anti-CD8 andanti-CD45RA antibodies and were labeled with carboxyfluoresceinsuccinimidyl ester (CFSE, 5 μm) according to manufacturer's protocol(Invitrogen). Next, they were seeded in 96 well-plates at aconcentration of 1.0×10⁵ cells/well. Cells were stimulated with solubleanti-CD3 mAb CLB-T3/4E (Pelicluster) at 10 μg/ml (used s/n of hybridomaculture), anti-CD28 mAb CLB-CD28/1 (Pelicluster) at 0.02 μg/ml, inpresence of hCD27.15 at 10 μg/ml or isotype control.

After culture for the indicated number of days, cells were counted usinga CASY cell counter (Scharfe System GmbH), viability was determined byusing propidium iodide (PI) and the number of cell divisions the T cellshad undergone was assessed by flow cytometric analysis of CFSEfluorescence intensity (FACS Calibur). FIG. 4 illustrates that hCD27.15is also promoting survival and proliferation of CD8⁺ cells.

hCD27.15 Stimulates CD8⁺ T Cells to Produce Specific Cytokines

Human naïve CD8⁺ T cells were purified and stimulated as indicatedabove. Culture supernatants were taken after 72 h of culture andanalyzed for cytokine secretion by 27-Plex Luminex according tomanufacturer's instructions (Biorad, cat. no. 171A11127). As shown inFIG. 5A, hCD27.15 induced the secretion of TNF-α, IL-2, IFN-γ, CXCL10,IL-13 and GM-CSF. In addition, cells were used to perform intracellularstaining for IL-2 and IFNγ. After 72 hours of culture, cells werecultured with PMA (conc) and ionomycin (conc) for 4 hours in thepresence of Golgi-Plug (1 μg/ml: BD Biosciences). The total number ofCD8⁺ cells is not dramatically different between hCD27.15 and nonestimulated cells indicating that the increased secretion of cytokines ismerely caused by a qualitative increase of cytokines per cell (FIG. 5B).

Example 4 hCD27.15 Antibody Sequences

Cloning of Immunoglobulin cDNAs

Degenerate primer PCR-based methods were used to determine the DNAsequences encoding the variable regions for the mouse antibody that isexpressed by hybridoma hCD27.15. Total RNA was isolated from 5×10⁶hybridoma cells using TRIZOL (Invitrogen), and gene specific cDNAs forthe heavy and light chains were synthesized using the M-MLV ReverseTranscriptase, RNase H Minus, point mutant kit (Promega, cat. no. M368C)according to the manufacturer's instructions. The V_(H) and V_(L) geneswere PCR-amplified using a Novagen-based Ig-primer set (Novagen, SanDiego, Calif.) and Taq polymerase (Invitrogen). All PCR products thatmatched the expected amplicon size of 500 bp were cloned into pCR4 TOPOvector (Invitrogen), and the constructs were transformed in One ShotCompetent Top10 E. coli (Invitrogen) according to the manufacturer'sinstructions.

Clones were screened by colony PCR using universal M13 forward andreverse primers, and at least two clones from each reaction wereselected for DNA sequencing analysis. CDRs were identified following theKabat rules (Kabat et al., 1991. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication No. 91-3242). The amino acidsequences were confirmed by mass spectrometry.

The sequences are disclosed in the attached Sequence Listing, FIG. 6 andlisted in Table 4.

TABLE 4 Sequence ID numbers for murine anti-human hCD27.15 antibody ofthis invention. SEQ ID NO: Description 1 hCD27.15 heavy chain variableregion (DNA) 2 hCD27.15 light chain variable region (DNA) 3 hCD27.15heavy chain variable region (AA) 4 hCD27.15 light chain variable region(AA) 5 hCD27.15 heavy chain CDR1 (AA) 6 hCD27.15 heavy chain CDR2 (AA) 7hCD27.15 heavy chain CDR3 (AA) 8 hCD27.15 light chain CDR1 (AA) 9hCD27.15 light chain CDR2 (AA) 10 hCD27.15 light chain CDR3 (AA)

The invention is further described by the following numbered paragraphs:

1. A binding compound, which binds the same epitope of human CD27 asmonoclonal antibody hCD27.15, produced by hybridoma hCD27.15 which wasdeposited with the ATCC in on Jun. 2, 2010 under number PTA-11008.

2. The binding compound of paragraph 1, comprising:

-   -   an antibody heavy chain variable region comprising at least one        CDR selected from the group consisting of SEQ ID NOs: 5, 6 and        7, or a variant of any of said sequences; and/or    -   an antibody light chain variable region comprising at least one        CDR selected from the group consisting of SEQ ID NOs: 8, 9 and        10, or a variant of any of said sequences.

3. The binding compound of paragraph 1 or 2, comprising:

-   -   a heavy chain variable region comprising the amino acid sequence        of SEQ ID NO: 3 and a light chain variable region comprising the        amino acid sequence of SEQ ID NO: 4.

4. The binding compound of paragraph 1 or 2, which binds to CD27 andcomprises:

-   -   an antibody heavy chain variable region comprising the CDRs of        SEQ ID NOs: 5, 6 and 7, or a variant of any of said sequences;        and/or    -   an antibody light chain variable region comprising the CDRs of        SEQ ID NOs: 8, 9 and 10, or a variant of any of said sequences.

5. The binding compound of any one of the paragraphs 1-4, wherein any ofsaid variant(s) comprise up to three amino acid modifications.

6. The binding compound of any one of the paragraphs 1-5, which compoundis monoclonal antibody hCD27.15 as produced by hybridoma hCD27.15(deposit accession number PTA-11008) or a humanized version thereof.

7. The binding compound of any of the preceding paragraphs, wherein thebinding compound:

-   -   binds human CD27 with a K_(D) of about 100 nM or lower; and    -   blocks binding of human CD27 to human CD70 with an IC₅₀ of about        10 nM or lower.

8. A binding compound which competes for a binding epitope on human CD27with any of the binding compounds of paragraphs 1-7, and has one or moreof the following characteristics:

-   -   binds human CD27 with a K_(D) of about 100 nM or lower;    -   binds to human CD27 with about the same K_(D) as an antibody        having a heavy chain comprising the amino acid sequence of SEQ        ID NO: 3 and a light chain comprising the amino acid sequence of        SEQ ID NO: 4;    -   blocks binding of human human CD27 to human CD70 with an IC₅₀ of        about 10 nM or lower.

9. The binding compound of any one of the paragraphs 1-8, which is

-   -   a chimeric antibody or a fragment thereof;    -   a human antibody or a fragment thereof;    -   a humanized antibody or a fragment thereof; or    -   an antibody fragment selected from the group consisting of Fab,        Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, bispecific mAb and a diabody.

10. An isolated polynucleotide encoding the binding compound of any oneof the paragraphs 1-9.

11. Isolated polynucleotide of paragraph 10, comprising SEQ ID NOs 1 and2, which encode the heavy and light chain of hCD27.15.

12. Expression vector comprising the polynucleotide of paragraph 10 or11.

13. Host cell comprising the expression vector of paragraph 11 or thepolynucleotide of paragraph 10 or 11.

14. A method of producing a binding compound of any one of theparagraphs 1-9, which method comprises:

a) culturing host cell comprising an expression vector that comprises apolynucleotide encoding a binding compound of the invention under thecontrol of suitable regulatory sequences in culture medium underconditions wherein the polynucleotide is expressed, thereby producingpolypeptides comprising the light and heavy chain variable regions; and

b) recovering the polypeptides from the host cell or culture medium.

15. Composition comprising a binding compound of any one of theparagraphs 1-9 in combination with a pharmaceutically acceptable carrieror diluent.

16. Composition of paragraph 15, further comprising another activecompound, in particular a therapeutically active compound, more inparticular an anti-cancer drug.

17. A binding compound of any one of the paragraphs 1-9 for use intherapy and diagnosis.

18. The binding compound of paragraph 17, wherein the therapy comprises

-   -   stimulation of proliferation and/or survival of CD27⁺ cells;    -   treatment of cancer; or    -   treatment of an autoimmune disease.

19. A binding compound of any one of the paragraphs 1-9 for use inflow-cytometry, Western blotting, enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A binding compound, which binds the same epitopeof human CD27 as monoclonal antibody hCD27.15, produced by hybridomahCD27.15 which was deposited with the ATCC on Jun. 2, 2010 under numberPTA-11008.
 2. The binding compound of claim 1, comprising: an antibodyheavy chain variable region comprising at least one CDR selected fromthe group consisting of SEQ ID NOs: 5, 6 and 7, or a variant of any ofsaid sequences; and/or an antibody light chain variable regioncomprising at least one CDR selected from the group consisting of SEQ IDNOs: 8, 9 and 10, or a variant of any of said sequences.
 3. The bindingcompound of claim 1, comprising: a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 3 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:
 4. 4.The binding compound of claim 1, which binds to CD27 and comprises: anantibody heavy chain variable region comprising the CDRs of SEQ ID NOs:5, 6 and 7, or a variant of any of said sequences; and/or an antibodylight chain variable region comprising the CDRs of SEQ ID NOs: 8, 9 and10, or a variant of any of said sequences.
 5. The binding compound ofany one of the claims 1-4, wherein any of said variant(s) may compriseup to three amino acid modifications.
 6. The binding compound of claim1, which compound is monoclonal antibody hCD27.15 as produced byhybridoma hCD27.15 (deposit accession number PTA-11008) or a humanizedversion thereof.
 7. The binding compound of claim 1, wherein the bindingcompound: binds human CD27 with a K_(D) of about 100 nM or lower; andblocks binding of human CD27 to human CD70 with an IC₅₀ of about 10 nMor lower.
 8. A binding compound which competes for a binding epitope onhuman CD27 with any of the binding compounds of claim 1, and has one ormore of the following characteristics: binds human CD27 with a K_(D) ofabout 100 nM or lower; binds to human CD27 with about the same K_(D) asan antibody having a heavy chain comprising the amino acid sequence ofSEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQID NO: 4; blocks binding of human human CD27 to human CD70 with an IC₅₀of about 10 nM or lower.
 9. The binding compound of claim 1, which is achimeric antibody or a fragment thereof; a human antibody or a fragmentthereof; a humanized antibody or a fragment thereof; or an antibodyfragment selected from the group consisting of Fab, Fab′, Fab′-SH, Fv,scFv, F(ab′)₂, bispecific mAb and a diabody.
 10. An isolatedpolynucleotide encoding the binding compound as claimed in claim
 1. 11.Isolated polynucleotide of claim 10, comprising SEQ ID NOs 1 and 2,which encode the heavy and light chain of hCD27.15.
 12. Expressionvector comprising the polynucleotide of claim
 10. 13. Host cellcomprising the expression vector of claim
 11. 14. Host cell comprisingthe polynucleotide of claim
 10. 15. A method of producing a bindingcompound as claimed in claim 1, which method comprises: a) culturinghost cell comprising an expression vector that comprises apolynucleotide encoding a binding compound of the invention under thecontrol of suitable regulatory sequences in culture medium underconditions wherein the polynucleotide is expressed, thereby producingpolypeptides comprising the light and heavy chain variable regions; andb) recovering the polypeptides from the host cell or culture medium. 16.Composition comprising a binding compound as claimed in claim 1 incombination with a pharmaceutically acceptable carrier or diluent. 17.Composition as claimed in claim 16, further comprising another activecompound, in particular a therapeutically active compound, more inparticular an anti-cancer drug.
 18. A binding compound as claimed claim1 for use in therapy and diagnosis.
 19. The binding compound as claimedin claim 18, wherein the therapy comprises stimulation of proliferationand/or survival of CD27⁺ cells; treatment of cancer; or treatment of anautoimmune disease.
 20. A binding compound as claimed in claim 1 for usein flow-cytometry, Western blotting, enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.